One (1) of the most challenging problems in molecular design is developing molecules that can bind protein surfaces and block protein-protein interactions. The ability to modulate protein-protein interactions in a directed fashion opens the possibility of probing and controlling biological systems through design. The long-term objective of this proposal is to use in vitro selection experiments to target protein surfaces, exploring fundamental aspects of protein recognition, structure, function, specificity, and catalysis. In vitro genetic approaches currently represent a powerful operational solution to the protein design problem. Previously, the PI has conceived, developed, and implemented mRNA-peptide and protein fusions (hereafter "mRNA display"), to design proteins using in vitro selection experiments. mRNA display provides important advantages relative to other in vitro and in vivo protein design strategies, such as the ability to examine very large libraries (>1013 individual sequences) in the absence of a living cell, with tight experimental control over binding and stringency. In this proposal, we will use G-protein linked signaling (specifically G protein a subunits and G protein-coupled receptors) as targets for ligand design. Our goal is to explore the biophysical chemistry of protein recognition, as it pertains to this important signaling pathway. Our specific aims are: 1) To develop class- and state-specific G protein-directed ligands. 2) To test the function of our G protein-directed ligands. 3) To explore the structure and function of peptide ligands targeting the Methuselah G protein-coupled receptor (GPCR). 4) To develop novel antibody mimetic ligands targeting chemokine GPCRs.